Small Molecule Xanthine Oxidase Inhibitors and Methods of Use

ABSTRACT

Small molecule xanthine oxidase inhibitors are provided, as well as methods for their use in treating gout or hyperuricemia.

CROSS-REFERENCE TO PRIORITY APPLICATION

This application claims priority to U.S. Provisional Application No.61/734,409, filed Dec. 7, 2012, which is incorporated herein byreference in its entirety.

BACKGROUND

Gout is caused by hyperuricemia, namely, abnormally high levels of uricacid in the blood. Gout is usually present as acute inflammatoryarthritis, as well as tophi, kidney stones, or urate nephropathy. Goutaffects 1-2% of adults in developed countries and represents the mostcommon case of inflammatory arthritis in men. In the United States,gouty arthritis accounts for millions of outpatient visits annually.Furthermore, gout and hyperuricemia are associated with chronic diseasessuch as hypertension, diabetes mellitus, metabolic syndrome, and renaland cardiovascular disease.

Xanthine oxidase (XO) is a form of a molybdoflavin protein, xanthineoxidoreductase (XOR). It plays an important role in the catabolism ofpurines in humans, as it catalyzes the oxidation of hypoxanthine toxanthine and then catalyzes the oxidation of xanthine to uric acid.Meanwhile, reactive oxygen species (ROS), including superoxide and H₂O₂,are generated during this process. Uric acid can serve as an antioxidantto prevent macromolecular damage by ROS. However, overproduction of uricacid can cause hyperuricemia and lead to gout and other diseases.Therefore, maintaining uric acid at normal levels represents animportant therapeutic goal for the prevention of gout and relateddisorders. For most patients with primary gout, overproduction of uricacid is the primary cause of hyperuricemia.

Currently, two drugs have been developed to treat gout. Allopurinol isthe most commonly used therapy for chronic gout and has been usedclinically for more than 40 years. Allopurinol lowers uric acidproduction by inhibiting XO activity, and is used as a first-lineurate-lowering phamacotherapy. Allopurinol, a structural isomer ofhypoxanthine, is hydroxylated by XO to oxypurinol, which coordinatestightly to the reduced form of the molybdenum center, replacing theMo—OH group of the native enzyme. Unfortunately, while rare, allopurinolhas life-threatening side effects such as a hypersensitivity syndromeconsisting of fever, skin rash, eosinophilia, hepatitis, and renaltoxicity, for which the mortality rate approaches 20%. It also causesStevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TENS),two life-threatening dermatological conditions. Febuxostat, a non-purinexanthine oxidase inhibitor, has been approved for the management of goutin Europe and the United States. Side effects associated with febuxostattherapy include elevated serum liver enzymes, nausea, diarrhea,arthralgia, headache, and rash. The drugs available for treatment andprevention of hyperuricemia and gout remain limited. Therefore, safe andeffective xanthine oxidase inhibitors are needed.

SUMMARY

Provided herein are small molecule xanthine oxidase inhibitors. Alsoprovided herein are methods for their use in treating gout orhyperuricemia. A class of xanthine oxidase inhibitors described hereinincludes compounds of the following structure:

and pharmaceutically acceptable salts thereof. In these compounds, R¹,R², R³, R⁴, and R⁵ are each independently selected from hydrogen,hydroxyl, nitro, cyano, fluoro, chloro, bromo, trifluoromethyl,sulfonyl, and aldehyde, wherein R¹, R², R³, R⁴, and R⁵ are notsimultaneously hydrogen. Examples of suitable xanthine oxidaseinhibitors as described herein include the following compounds:

A xanthine oxidase inhibitor suitable for the methods described hereinalso includes the following compound:

and pharmaceutically acceptable salts thereof.

Also provided herein are methods for treating gout or hyperuricemia in asubject. A method of treating gout or hyperuricemia in a subjectincludes administering to the subject an effective amount of a xanthineoxidase inhibitor as described herein. Optionally, the methods fortreating gout or hyperuricemia in a subject can further includeadministering a second therapeutic agent, such as an anti-gout agent(e.g., allopurinol, benzbromarone, colchicine, probenecid, orsulfinpyrazone), an anti-inflammatory agent, or an antioxidant, to thesubject. Optionally, the compound is administered orally to the subject.

Further provided herein are methods for reducing uric acid productionand/or reactive oxygen species production in a subject. The methodsinclude administering to the subject an effective amount of a xanthineoxidase inhibitor as described herein. Optionally, the methods forreducing uric acid production and/or reactive oxygen species productionfurther comprise selecting a subject having gout or hyperuricemia.

Methods of inhibiting xanthine oxidase activity in a cell are alsoprovided herein. The methods include contacting a cell with an effectiveamount of a xanthine oxidase inhibitor as described herein. Optionally,the contacting is performed in vivo. Optionally, the contacting isperformed in vitro.

The details of one or more embodiments are set forth in the drawings andthe description below. Other features, objects, and advantages will beapparent from the description and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1A is a graph showing the inhibition of xanthine oxidase (XO)activity by test compounds (DH6NB, DHBA, THB-CHO, and DHNB) and acontrol compound (allopurinol). FIG. 1B is a Dixon Plot for DHNB atvarying concentrations of xanthine. FIG. 1C is a graph showing theeffects of pH on the inhibitory effect of DHNB on XO.

FIG. 2 is a graph comparing the xanthine oxidase inhibitory effects ofcatechol compounds, each at a concentration of 20 μM. The controlrepresents no inhibitor added.

FIG. 3 is a graph showing the time course of inhibition of xanthineoxidase activity by DHNB and allopurinol. XO activity was determinedunder standard conditions and started by adding 20 nM XO (open symbols)or by adding 50 μM xanthine following 4 min pre-incubation of XO andinhibitor (solid symbols). Circles, control—no inhibitor added; squares,with 6.67 μM allopurinol; triangle, with 6.67 μM DHNB.

FIG. 4 is a graph showing the influence of pre-incubation of 20 μMinhibitors with 20 nM XO on XO activity. Vanillin, DHB-CHO, DH6NB,THB-CHO, allopurinol (at 3.3 μM), and DHNB (at 6.6 μM and 20 μM) werethe tested compounds.

FIG. 5 is a graph demonstrating that DHNB inhibition of XO is notreversible by reducing agents. XO (10 mU/ml) and 20 μM DHNB werepre-incubated for 10 min at 25° C. in phosphate buffer (100 mM pH 7.4).Then a high level of GSH (20 mM), 2-mercaptoethanol (2-ME, 20 mM), ordithiothreitol (DTT, 20 mM) was added for 15 min, and XO activity wasanalyzed by the production of uric acid. A “+” signifies the addition ofthe reagent DHNB, GSH, 2-ME, and/or DTT. A “−” signifies that thereagent was not added. Data represent the mean±S.E. at least threeindependent determinations.

FIG. 6A is a graph of the time course of absorption change of DHNB (327nm, arrow indicates decrease) and the formation of the product (279 nm,arrow indicates increase) following the mixing of 30 nM XO and 30 μMDHNB in 0.1M phosphate buffer (pH 7.4). FIG. 6B is a graph showing theeffects of pH on the conversion of DHNB by XO enzyme. FIG. 6C containsan HPLC profile of DHNB (control) and a DHNB/XO mixture after incubationfor 3 days. FIG. 6D is a MS/MS spectrum of DHNB. FIG. 6E is a MS/MSspectrum of the DHNB/XO product, DHNB-COOH.

FIG. 7 contains graphs showing the antioxidant activities of DHNB,DH6NB, DHBA, DHB-CHO, THB-CHO, and allopurinol on the scavenging of freeradical DPPH (panel A), hypochlorous acid (HOCl) (panel B),peroxynitrite (ONOO⁻) (panel C), and/or superoxide ion (O₂ ^(−)) (panelD). Vitamin C (Vit C) or glutathione (GSH) was used as a control. Eachcompound was used at a concentration of 20 μM.

FIG. 8 is a graph showing the concentration dependent DPPH scavengingactivities of DHNB, DHBA, DHB-CHO, and allopurinol. Vitamin C (Vit C)and Vitamin E (Vit E) were used as controls.

FIG. 9 is a graph showing the concentration dependent HOCl scavengingactivities of DHNB, caffeic acid (CA), DHBA, DHB-CHO, DHB-COOH, andallopurinol. Vitamin C (Vit C) was used as a control.

FIG. 10 is a graph showing the concentration dependent peroxynitritescavenging activity of DHNB, DHBA, DHB-CHO, DH6NB, DMB-CH₂OH, caffeicacid, THB-CHO, gallic acid, DHB-COOH, and vanillin. Vitamin C (Vit C)was used as a control.

FIG. 11 is a graph showing the concentration dependent superoxide ionscavenging activity of caffeic acid and DHBA. Glutathione (GSH) was usedas a control.

FIG. 12 is a graph showing the dose dependent hyperuricemic effects ofallantoxanamide in mice.

FIG. 13 is a graph showing the time course of the hypouricemic effect ofDHNB and allopurinol on the allantoxanamide induced hyperuricemic mice.

FIG. 14A is a photograph of allopurinol treated mice at 2.5 weeks. FIG.14B is a photograph of allopurinol treated mice at 3 weeks. FIG. 14C isa photograph of allopurinol treated mice at 4 weeks. FIG. 14D is aphotograph of allopurinol treated mice at 6 weeks. FIG. 14E is aphotograph of DHNB treated mice at 4 weeks.

FIGS. 15A and 15B are graphs showing the inhibitory effects of DNSA andNHBA, respectively, on XO activity by measuring the initial rate of uricacid formation.

FIG. 16 is a graph comparing the xanthine oxidase inhibitory effects ofcatechol compounds at a concentration of 20 μM. The control representsno inhibitor added.

FIG. 17 is a graph demonstrating the influence of pre-incubation of DNSAwith XO on XO activity.

FIG. 18 is a graph demonstrating XO activity after pre-incubation withinhibitors DNSA and DHNB for 20 hours.

DETAILED DESCRIPTION

Provided herein are small molecule xanthine oxidase inhibitors andmethods for their use in treating gout or hyperuricemia in a subject.The xanthine oxidase inhibitors are administered in an effective amountto treat gout or hyperuricemia in a subject.

I. Compounds

A class of xanthine oxidase inhibitors described herein is representedby Formula

and pharmaceutically acceptable salts thereof.

In Formula I, R¹, R², R³, R⁴, and R⁵ are each independently selectedfrom hydrogen, hydroxyl, nitro, cyano, fluoro, chloro, bromo,trifluoromethyl, sulfonyl, and aldehyde (i.e., —CHO). Optionally, thesulfonyl is methylsulfonyl or sulfonic acid.

Also, in Formula I, R¹, R², R³, R⁴, and R⁵ are not simultaneouslyhydrogen.

Examples of Formula I include the following compounds:

An additional xanthine oxidase inhibitor useful with the methodsdescribed herein includes the following compound:

and pharmaceutically acceptable salts thereof.

II. Pharmaceutical Formulations

The compounds described herein or derivatives thereof can be provided ina pharmaceutical composition. Depending on the intended mode ofadministration, the pharmaceutical composition can be in the form ofsolid, semi-solid or liquid dosage forms, such as, for example, tablets,suppositories, pills, capsules, powders, liquids, or suspensions,preferably in unit dosage form suitable for single administration of aprecise dosage. The compositions will include a therapeuticallyeffective amount of the compound described herein or derivatives thereofin combination with a pharmaceutically acceptable carrier and, inaddition, may include other medicinal agents, pharmaceutical agents,carriers, or diluents. By pharmaceutically acceptable is meant amaterial that is not biologically or otherwise undesirable, which can beadministered to an individual along with the selected compound withoutcausing unacceptable biological effects or interacting in a deleteriousmanner with the other components of the pharmaceutical composition inwhich it is contained.

As used herein, the term carrier encompasses any excipient, diluent,filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, orother material well known in the art for use in pharmaceuticalformulations. The choice of a carrier for use in a composition willdepend upon the intended route of administration for the composition.The preparation of pharmaceutically acceptable carriers and formulationscontaining these materials is described in, e.g., Remington'sPharmaceutical Sciences, 21st Edition, ed. University of the Sciences inPhiladelphia, Lippincott, Williams & Wilkins, Philadelphia Pa., 2005.Examples of physiologically acceptable carriers include buffers, such asphosphate buffers, citrate buffer, and buffers with other organic acids;antioxidants including ascorbic acid; low molecular weight (less thanabout 10 residues) polypeptides; proteins, such as serum albumin,gelatin, or immunoglobulins; hydrophilic polymers, such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates, including glucose, mannose, or dextrins; chelatingagents, such as EDTA; sugar alcohols, such as mannitol or sorbitol;salt-forming counterions, such as sodium; and/or nonionic surfactants,such as TWEEN® (ICI, Inc.; Bridgewater, N.J.), polyethylene glycol(PEG), and PLURONICS™ (BASF; Florham Park, N.J.).

Compositions containing the compounds described herein or derivativesthereof suitable for parenteral injection may comprise physiologicallyacceptable sterile aqueous or nonaqueous solutions, dispersions,suspensions or emulsions, and sterile powders for reconstitution intosterile injectable solutions or dispersions. Examples of suitableaqueous and nonaqueous carriers, diluents, solvents or vehicles includewater, ethanol, polyols (propyleneglycol, polyethyleneglycol, glycerol,and the like), suitable mixtures thereof, vegetable oils (such as oliveoil) and injectable organic esters such as ethyl oleate. Proper fluiditycan be maintained, for example, by the use of a coating such aslecithin, by the maintenance of the required particle size in the caseof dispersions and by the use of surfactants.

These compositions may also contain adjuvants, such as preserving,wetting, emulsifying, and dispensing agents. Prevention of the action ofmicroorganisms can be promoted by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, sorbic acid, andthe like. Isotonic agents, for example, sugars, sodium chloride, and thelike may also be included. Prolonged absorption of the injectablepharmaceutical form can be brought about by the use of agents delayingabsorption, for example, aluminum monostearate and gelatin.

Solid dosage forms for oral administration of the compounds describedherein or derivatives thereof include capsules, tablets, pills, powders,and granules. In such solid dosage forms, the compounds described hereinor derivatives thereof is admixed with at least one inert customaryexcipient (or carrier), such as sodium citrate or dicalcium phosphate,or (a) fillers or extenders, as for example, starches, lactose, sucrose,glucose, mannitol, and silicic acid, (b) binders, as for example,carboxymethylcellulose, alignates, gelatin, polyvinylpyrrolidone,sucrose, and acacia, (c) humectants, as for example, glycerol, (d)disintegrating agents, as for example, agar-agar, calcium carbonate,potato or tapioca starch, alginic acid, certain complex silicates, andsodium carbonate, (e) solution retarders, as for example, paraffin, (f)absorption accelerators, as for example, quaternary ammonium compounds,(g) wetting agents, as for example, cetyl alcohol, and glycerolmonostearate, (h) adsorbents, as for example, kaolin and bentonite, and(i) lubricants, as for example, talc, calcium stearate, magnesiumstearate, solid polyethylene glycols, sodium lauryl sulfate, or mixturesthereof. In the case of capsules, tablets, and pills, the dosage formsmay also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethyleneglycols, andthe like.

Solid dosage forms such as tablets, dragees, capsules, pills, andgranules can be prepared with coatings and shells, such as entericcoatings and others known in the art. They may contain opacifying agentsand can also be of such composition that they release the activecompound or compounds in a certain part of the intestinal tract in adelayed manner. Examples of embedding compositions that can be used arepolymeric substances and waxes. The active compounds can also be inmicro-encapsulated form, if appropriate, with one or more of theabove-mentioned excipients.

Liquid dosage forms for oral administration of the compounds describedherein or derivatives thereof include pharmaceutically acceptableemulsions, solutions, suspensions, syrups, and elixirs. In addition tothe active compounds, the liquid dosage forms may contain inert diluentscommonly used in the art, such as water or other solvents, solubilizingagents, and emulsifiers, as for example, ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propyleneglycol, 1,3-butyleneglycol, dimethylformamide, oils,in particular, cottonseed oil, groundnut oil, corn germ oil, olive oil,castor oil, sesame oil, glycerol, tetrahydrofurfuryl alcohol,polyethyleneglycols, and fatty acid esters of sorbitan, or mixtures ofthese substances, and the like.

Besides such inert diluents, the composition can also include additionalagents, such as wetting, emulsifying, suspending, sweetening, flavoring,or perfuming agents.

Suspensions, in addition to the active compounds, may contain additionalagents, as for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, or mixtures of thesesubstances, and the like.

Compositions of the compounds described herein or derivatives thereoffor rectal administrations are optionally suppositories, which can beprepared by mixing the compounds with suitable non-irritating excipientsor carriers, such as cocoa butter, polyethyleneglycol or a suppositorywax, which are solid at ordinary temperatures but liquid at bodytemperature and, therefore, melt in the rectum or vaginal cavity andrelease the active component.

Dosage forms for topical administration of the compounds describedherein or derivatives thereof include ointments, powders, sprays, andinhalants. The compounds described herein or derivatives thereof areadmixed under sterile conditions with a physiologically acceptablecarrier and any preservatives, buffers, or propellants as may berequired. Ophthalmic formulations, ointments, powders, and solutions arealso contemplated as being within the scope of the compositions.

The compositions can include one or more of the compounds describedherein and a pharmaceutically acceptable carrier. As used herein, theterm pharmaceutically acceptable salt refers to those salts of thecompound described herein or derivatives thereof that are, within thescope of sound medical judgment, suitable for use in contact with thetissues of subjects without undue toxicity, irritation, allergicresponse, and the like, commensurate with a reasonable benefit/riskratio, and effective for their intended use, as well as the zwitterionicforms, where possible, of the compounds described herein. The term saltsrefers to the relatively non-toxic, inorganic and organic acid additionsalts of the compounds described herein. These salts can be prepared insitu during the isolation and purification of the compounds or byseparately reacting the purified compound in its free base form with asuitable organic or inorganic acid and isolating the salt thus formed.Representative salts include the hydrobromide, hydrochloride, sulfate,bisulfate, nitrate, acetate, oxalate, valerate, oleate, palmitate,stearate, laurate, borate, benzoate, lactate, phosphate, tosylate,citrate, maleate, fumarate, succinate, tartrate, naphthylate mesylate,glucoheptonate, lactobionate, methane sulphonate, and laurylsulphonatesalts, and the like. These may include cations based on the alkali andalkaline earth metals, such as sodium, lithium, potassium, calcium,magnesium, and the like, as well as non-toxic ammonium, quaternaryammonium, and amine cations including, but not limited to ammonium,tetramethylammonium, tetraethylammonium, methylamine, dimethylamine,trimethylamine, triethylamine, ethylamine, and the like. (See S. M.Barge et al., J. Pharm. Sci. (1977) 66, 1, which is incorporated hereinby reference in its entirety, at least, for compositions taughttherein.) Administration of the compounds and compositions describedherein or pharmaceutically acceptable salts thereof can be carried outusing therapeutically effective amounts of the compounds andcompositions described herein or pharmaceutically acceptable saltsthereof as described herein for periods of time effective to treat adisorder. The effective amount of the compounds and compositionsdescribed herein or pharmaceutically acceptable salts thereof asdescribed herein may be determined by one of ordinary skill in the artand includes exemplary dosage amounts for a mammal of from about 0.5 toabout 200 mg/kg of body weight of active compound per day, which may beadministered in a single dose or in the form of individual divideddoses, such as from 1 to 4 times per day. Alternatively, the dosageamount can be from about 0.5 to about 150 mg/kg of body weight of activecompound per day, about 0.5 to 100 mg/kg of body weight of activecompound per day, about 0.5 to about 75 mg/kg of body weight of activecompound per day, about 0.5 to about 50 mg/kg of body weight of activecompound per day, about 0.5 to about 25 mg/kg of body weight of activecompound per day, about 1 to about 20 mg/kg of body weight of activecompound per day, about 1 to about 10 mg/kg of body weight of activecompound per day, about 20 mg/kg of body weight of active compound perday, about 10 mg/kg of body weight of active compound per day, or about5 mg/kg of body weight of active compound per day. Those of skill in theart will understand that the specific dose level and frequency of dosagefor any particular subject may be varied and will depend upon a varietyof factors, including the activity of the specific compound employed,the metabolic stability and length of action of that compound, thespecies, age, body weight, general health, sex and diet of the subject,the mode and time of administration, rate of excretion, drugcombination, and severity of the particular condition.

III. Methods of Making the Compounds

The compounds described herein can be prepared in a variety of waysknown to one skilled in the art of organic synthesis or variationsthereon as appreciated by those skilled in the art. The compoundsdescribed herein can be prepared from readily available startingmaterials. Optimum reaction conditions may vary with the particularreactants or solvents used, but such conditions can be determined by oneskilled in the art.

Variations on Formula I and the compounds described herein include theaddition, subtraction, or movement of the various constituents asdescribed for each compound. Similarly, when one or more chiral centersare present in a molecule, the chirality of the molecule can be changed.Additionally, compound synthesis can involve the protection anddeprotection of various chemical groups. The use of protection anddeprotection, and the selection of appropriate protecting groups can bedetermined by one skilled in the art. The chemistry of protecting groupscan be found, for example, in Wuts and Greene, Protective Groups inOrganic Synthesis, 4th Ed., Wiley & Sons, 2006, which is incorporatedherein by reference in its entirety.

Reactions to produce the compounds described herein can be carried outin solvents, which can be selected by one of skill in the art of organicsynthesis. Solvents can be substantially nonreactive with the startingmaterials (reactants), the intermediates, or products under theconditions at which the reactions are carried out, i.e., temperature andpressure. Reactions can be carried out in one solvent or a mixture ofmore than one solvent. Product or intermediate formation can bemonitored according to any suitable method known in the art. Forexample, product formation can be monitored by spectroscopic means, suchas nuclear magnetic resonance spectroscopy (e.g., ¹H or ¹³C) infraredspectroscopy, spectrophotometry (e.g., UV-visible), or massspectrometry, or by chromatography such as high performance liquidchromatography (HPLC) or thin layer chromatography.

Optionally, the compounds described herein can be obtained fromcommercial sources. The compounds can be obtained from, for example,Sigma Chemical Co. (St. Louis, Mo.), VWR International (Radnor, Pa.), orOakwood Products, Inc. (West Columbia, S.C.).

IV. Methods of Use

Provided herein are methods of treating or preventing gout andhyperuricemia in a subject. The methods include administering to asubject an effective amount of one or more of the compounds orcompositions described herein, or a pharmaceutically acceptable saltthereof. The expression “effective amount,” when used to describe anamount of compound in a method, refers to the amount of a compound thatachieves the desired pharmacological effect or other effect, forexample, an amount that results in uric acid production reduction. Thecompounds and compositions described herein or pharmaceuticallyacceptable salts thereof are useful for treating gout or hyperuricemiain humans, including, without limitation, pediatric and geriatricpopulations, and in animals, e.g., veterinary applications. Optionally,the methods are used to treat conditions associated with elevated uricacid levels, including chronic gouty arthritis, acute inflammatoryarthritis, uric acid nephropathy, kidney stones, or tophi.

Further described herein are methods of reducing uric acid productionand/or reactive oxygen species production in a subject. The methodsinclude administering to the subject one or more of the compounds asdescribed herein. Optionally, the methods can further comprise selectinga subject having gout or hyperuricemia.

The methods described herein can further comprise administering to thesubject a second therapeutic agent. Thus, the provided compositions andmethods can include one or more additional agents. The one or moreadditional agents and the compounds described herein or pharmaceuticallyacceptable salts thereof can be administered in any order, includingconcomitant, simultaneous, or sequential administration. Sequentialadministration can be temporally spaced order of up to several daysapart. The methods can also include more than a single administration ofthe one or more additional agents and/or the compounds described hereinor pharmaceutically acceptable salts or prodrugs thereof. Theadministration of the one or more additional agents and the compoundsdescribed herein or pharmaceutically acceptable salts or prodrugsthereof can be by the same or different routes and concurrently orsequentially.

Therapeutic agents include, but are not limited to, anti-gout agents.For example, the anti-gout agent can be allopurinol, benzbromarone,colchicine, probenecid, or sulfinpyrazone. Therapeutic agents alsoinclude anti-inflammatory agents. Examples of suitable anti-inflammatoryagents include, for example, steroidal and nonsteroidalanti-inflammatory drugs (e.g., ibuprofen and prednisone). Thetherapeutic agent can also be, for example, an antioxidant. Examples ofsuitable antioxidants include, for example, α-tocopherol, beta-carotene,butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), caffeicacid, lutein, lycopene, selenium, tert-butylhydroquinone (TBHQ), VitaminA, Vitamin C, and Vitamin E. Further examples of suitable antioxidantsinclude putative antioxidant botanticals, such as, for example, grapeseeds, green tea, Scutellaria baicalensis, American ginseng, ginkgobiloba, and the like.

Any of the aforementioned therapeutic agents can be used in anycombination with the compositions described herein. Combinations areadministered either concomitantly (e.g., as an admixture), separatelybut simultaneously (e.g., via separate intravenous lines into the samesubject), or sequentially (e.g., one of the compounds or agents is givenfirst followed by the second). Thus, the term combination is used torefer to concomitant, simultaneous, or sequential administration of twoor more agents.

The methods and compounds as described herein are useful for bothprophylactic and therapeutic treatment. For prophylactic use, atherapeutically effective amount of the compounds and compositions orpharmaceutically acceptable salts thereof as described herein areadministered to a subject prior to onset (e.g., before obvious signs ofgout or hyperuricemia), during early onset (e.g., upon initial signs andsymptoms of gout or hyperuricemia), or after the development of gout orhyperuricemia. Prophylactic administration can occur for several days toyears prior to the manifestation of symptoms of gout or hyperuricemia.Therapeutic treatment involves administering to a subject atherapeutically effective amount of the compounds and compositions orpharmaceutically acceptable salts thereof as described herein after goutor hyperuricemia is diagnosed.

The methods and compounds described herein are also useful in inhibitingxanthine oxidase activity in a cell. The methods include contacting acell with an effective amount of a xanthine oxidase inhibitor asdescribed herein. Optionally, the contacting is performed in vivo.Optionally, the contacting is performed in vitro.

V. Kits

Also provided herein are kits for treating or preventing gout orhyperuricemia in a subject. A kit can include any of the compounds orcompositions described herein. For example, a kit can include a compoundof Formula I or any of the compounds described herein. A kit can furtherinclude one or more additional agents, such as anti-gout agents (e.g.,allopurinol, benzbromarone, colchicine, probenecid, or sulfinpyrazone),anti-inflammatory agents, or antioxidants. A kit can include an oralformulation of any of the compounds or compositions described herein. Akit can additionally include directions for use of the kit (e.g.,instructions for treating a subject), a container, a means foradministering the compounds or compositions, and/or a carrier.

As used herein the terms treatment, treat, or treating refer to a methodof reducing one or more symptoms of a disease or condition. Thus in thedisclosed method, treatment can refer to a 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, or 100% reduction in the severity of one or more symptomsof the disease or condition. For example, a method for treating adisease is considered to be a treatment if there is a 10% reduction inone or more symptoms or signs of the disease in a subject as compared toa control. As used herein, control refers to the untreated condition.Thus the reduction can be a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,100%, or any percent reduction in between 10% and 100% as compared tonative or control levels. It is understood that treatment does notnecessarily refer to a cure or complete ablation of the disease,condition, or symptoms of the disease or condition.

As used herein, the terms prevent, preventing, and prevention of adisease or disorder refer to an action, for example, administration of acomposition or therapeutic agent, that occurs before or at about thesame time a subject begins to show one or more symptoms of the diseaseor disorder, which inhibits or delays onset or severity of one or moresymptoms of the disease or disorder.

As used herein, references to decreasing, reducing, or inhibitinginclude a change of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% orgreater as compared to a control level. Such terms can include, but donot necessarily include, complete elimination.

As used herein, subject means both mammals and non-mammals. Mammalsinclude, for example, humans; non-human primates, e.g., apes andmonkeys; cattle; horses; sheep; rats; mice; pigs; and goats. Non-mammalsinclude, for example, fish and birds.

Throughout this application, various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application.

EXAMPLES

The following examples are set forth below to illustrate the methods andresults according to the disclosed subject matter. These examples arenot intended to be inclusive of all aspects of the subject matterdisclosed herein, but rather to illustrate representative methods andresults. These examples are not intended to exclude equivalents andvariations of the subject matter described herein which are apparent toone skilled in the art.

Efforts have been made to ensure accuracy with respect to numbers (e.g.,amounts, temperature, etc.) but some errors and deviations should beaccounted for. Unless indicated otherwise, parts are parts by weight,temperature is in ° C. or is at ambient temperature, and pressure is ator near atmospheric. There are numerous variations and combinations ofreaction conditions, e.g., component concentrations, temperatures,pressures, and other reaction ranges and conditions that can be used tooptimize the product purity and yield obtained from the describedprocess. Only reasonable and routine experimentation will be required tooptimize such process conditions.

Xanthine oxidase from bovine milk, xanthine, allopurinol,3,4-dihydroxybenzaldehyde, phosphate buffered saline (PBS) solution,potassium nitrite (KNO₂), dioxide manganese (MnO₂),diethylene-triamine-pentaacetic acid (DTPA), EDTA, ferrous ammoniumsulfate, hydrogen peroxide (H₂O₂), sodium hypochlorite, DPPH,5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB), sodium borohydride,potassium persulfate, ascorbic acid, and (±)-α-tocopherol were obtainedfrom Sigma Chemical Co. (St. Louis, Mo.).3,4-Dihydroxy-5-nitrobenzaldehyde, 3,4-dimethoxybenzyl alcohol,3,4-dihydroxyphenyl ethanol, caffeic acid, 3,4-dihydroxyphenyl ethanol,3,4,5-trihydroxybenzaldehyde hydrate, 4-hydroxy-3-methoxybenzyl alcohol,and 3,4-dihydroxybenzoic acid were obtained from VWR International(Radnor, Pa.). 3,4-Dihydroxy-6-nitrobenzaldehyde was obtained fromOakwood Products, Inc. (West Columbia, S.C.).

Data are presented as mean±SD as compared to the negative control.Statistical significance was determined by a Student's t-test (twotailed). A value of P<0.05 was considered significant.

Example 1 XO Inhibition Assay

XO activity was determined by the method of continuousspectrophotometric rate determination by monitoring the increase ofabsorption at 295 nm of uric acid in 67 mM phosphate buffer (pH 7.4)containing 20 nM xanthine oxidase with an activity of 5 mU/ml, with orwithout the compounds as described herein. After pre-incubation for 1 to5 min at 25° C., the formation of uric acid in the reaction mixture wasinitiated by the addition of 50 μM xanthine. The test compounds andpositive control are shown in Scheme 1. Allopurinol was used as apositive control. All compounds, including allopurinol, were dissolvedin H₂O or an aqueous solution. H₂O was used as the negative control.

XO inhibition—The inhibitory activity of xanthine oxidase by DHNB, DHBA,DH6NB and THB-CHO was determined in vitro by the formation of uric acid,which was measured spectrophotometrically by following the increase inabsorbance of uric acid at 295 nm. When 20 nM XO was mixed withincreasing concentrations of allopurinol, DHNB, DH6NB, DHBA, or THB-CHO(Scheme 1), the initial rate of uric acid formation showed aconcentration-dependent decrease compared to the control, reflecting thedecrease of XO activity (see FIG. 1A). DHNB significantly inhibited XOactivity with an IC₅₀ value of 3 μM, which is close to allopurinol'svalue of 1.8 μM. The IC₅₀ values for DHBA and DH6NB were 76 and 96 μM,respectively, indicating weak inhibition of XO activity. The IC₅₀ valuefor THB-CHO was too high to determine. After DHNB and XO werepre-incubated for 1 min, xanthine was added to initiate the reaction.The initial rate of uric acid formation did not change with increasingconcentrations of xanthine. A Dixon plot of a steady-state kinetic studyof DHNB inhibition on XO activity indicated that the initial rates didnot change with increasing xanthine concentrations when theconcentration of DHNB was fixed (see FIG. 1B). The pH dependence of DHNBinhibition indicated that neutral or slightly acidic solutions benefitthe inhibition (see FIG. 1C).

Structure activity relationship of XO inhibition—The inhibition of XOactivity by the other compounds shown in Scheme 1, including a drugentacapone, was also studied. These compounds possess the same catecholskeleton in their structures but with different functional groups. Theability of each compound to inhibit XO at a concentration of 20 μM wascompared with that of allopurinol (see FIG. 2). Although these compoundshave similar structures, their XO inhibition capacities were different.Compounds containing a —CHO group, such as DHNB, DH6NB, DHB-CHO andTHB-CHO, had inhibitory effects on XO. Vanillin, although containing a—CHO group, had no inhibition on XO activity. DHBA has no —CHO group butit showed moderate inhibition. Other compounds, such as DHB-COOH, gallicacid, caffeic acid, hydroxytyrosol, DMB-CH₂OH and DHNB-CH₂OH, containing—COOH or —CH₂OH, had no effect on XO.

Entacapone, the catechol-O-methyl transferase (COMT) inhibitor, did notshow any inhibitory effect on XO even though it possesses a3,4-dihydroxy-5-nitrobenzyl moiety, as does DHNB, the strong XOinhibitor.

XO inhibition of DHNB is irreversible—DHNB displayed time-dependentinhibition of XO activity, similar to that of allopurinol. When XO (20nM) was added to the mixture of xanthine (50 μM) and the inhibitor (6.67μM) to start the reaction, both DHNB and allopurinol showedtime-dependent inhibition (see FIG. 3). An excess of 6.67 μM DHNB or6.67 μM allopurinol reduced the rate gradually and finally reached asteady state level of catalytic activity. No complete inactivation wasobserved at the tested condition. After pre-incubation of 20 nM XO with10 μM DHNB or 10 μM allopurinol for 4 min, the catalytic activity of XOdisplayed a steady state from the beginning and both DHNB andallopurinol showed a similar inhibitory pattern. Furthermore, afterpre-incubation of the DHNB and XO for 0 to 5 min at 25° C., theformation of uric acid in the reaction mixture was initiated by theaddition of 50 μM xanthine. Pre-incubation of the DHNB and XOsignificantly increased the inhibition. For example, 6.67 μM DHNB onlyinhibited 20% of XO activity without pre-incubation, as determined bycomparing with the control of the initial rate in the first 200 s.However, after a 2-min incubation of DHNB and XO, the inhibition wasincreased to 75%. Meanwhile, pre-incubation also affected the XOinhibition of DH6NB, DHB-CHO, and THB-CHO (see FIG. 4). The inhibitionof DHB-CHO on XO activity was not concentration dependent but insteadwas pre-incubation time dependent. However, pre-incubation of XO andDHBA did not increase the inhibition potency of DHBA. For othercompounds listed in Scheme 1, such as vanillin, DHB-COOH,hydroxytyrosol, DMB-CH₂OH, THB-CH₂OH, gallic acid, DHNB-CH₂OH, caffeicacid, and entacapone, pre-incubation with XO for up to 5 min did notshow any inhibitory effect. These results indicate that DHNB is anirreversible XO inhibitor in the tested condition. Also, XO was treatedwith 20 μM DHNB to induce inhibition; the reaction mixture was thentreated with high levels of glutathione (GSH; 20 mM), dithiothreitol(DTT; 20 mM) or 2-mercaptoethanol (2-ME; 20 mM), which did not abolishthe inhibition (see FIG. 5).

Example 2 Conversion of DHNB to Products by XO

The reaction kinetics of DHNB with XO at different pH values weremeasured using a spectrophotometer by monitoring the decay of DHNB at327 nm in a system of 30 nM XO with 30 μM DHNB in phosphate buffer withpH 6.5 to 8.5. The extinction coefficient of DHNB at 327 nm was measuredas 15,600 M⁻¹ cm⁻¹. The sample for product analysis by mass spectroscopyand HPLC was prepared by mixing 0.3 U XO with 4 mg DHNB in 1 mLphosphate buffer (pH 7.4) for 3 days. The DHNB/XO samples were analyzedby HPLC (Bio-Rad BioLogic DuoFlow; Hercules, Calif.) equipped with a250×4.6 mm, 5 micron Phenomenex C-18 (2) Luna column, with a mobilephase of 40% acetonitrile/water. DHNB and its product were monitored bythe optical absorption at 279 nm and 327 nm.

Negative electrospray ionization-mass spectrometry (ESI-MS) and tandem(MS-MS) were applied to detect and confirm the reaction products of DHNBwith XO. All mass spectrometric experiments were performed on an API3200-Qtrap triple quadrupole mass spectrometer (Applied Biosystem/MDSSCIEX; Foster City, Calif.) equipped with a TurbolonSpray™ source. Themain working parameters for mass spectrum were set as follows: ion-sprayvoltage, −4.5 kV; ion source temperature, 600° C.; gas 1, 40 psi; gas 2,40 psi; curtain gas, 20 psi; collision gas, high.

Reaction of XO and DHNB—To determine how DHNB inhibits XO enzymeactivity, 30 μM DHNB was incubated with 15 mU/ml (or 30 nM) XO inphosphate buffer (pH 7.4) and xanthine was then added to initiate thereaction as discussed above. The inhibition of DHNB on XO activitylasted up to 20 h. After that, the enzymatic activity of XO wasrecovered. The optical spectral change of DHNB was measured in a systemwithout xanthine, i.e., 30 μM DHNB with 15 mU/ml (or 30 nM) XO inphosphate buffer. The absorption of DHNB at 327 nm decreased with timeand a new peak appeared at 270 nm (see FIG. 6A). The decay rate was inthe range of 10⁻¹⁰ mol/L/s and was pH dependent, i.e., the higher the pHvalue, the faster DHNB decayed (see FIG. 6B). Without XO, however, DHNBitself was very stable. At room temperature, DHNB was converted by XOenzyme to a product which has no inhibitory effect on XO and thusrecovered the XO activity as the concentration of DHNB decreased. TheUV-VIS spectrum of the product was different from that of DHNB. HPLCanalysis of DHNB/XO showed a new peak which was more polar than DHNB(see FIG. 6C). Mass spectrometric analysis of the product gave amolecular ion ([M-H]⁻) peak at m/z 198 in the EI mass spectrum, whileDHNB showed [M-H]⁻ peak at m/z 182 (see FIGS. 6D, E). The MS/MS of m/z182 of DHNB gave several typical fragments such as m/z 165 ([M-H—OH]⁻),152 ([M-H—CHO—H]⁻) and 135 (m/z 152-OH). However, the MS/MS of molecularion at m/z 198 of the product gave a first main fragment at m/z 154, amass difference of 44 indicating a loss of CO₂, which further loses a—OH to give a fragment at m/z 137. Based on the mass spectrum, theproduct is 3,4-dihydroxy-5-nitrobenzoic acid, implying that DHNB isoxidized to the acid by the enzyme.

Example 3 Antioxidant Activity of DHNB

In addition, unlike allopurinol, the compounds described herein canserve as antioxidants. This was determined by testing the ability of thecompounds to scavenge DPPH, HOCl, peroxynitrite, and the superoxide ion.Each experiment was performed three times and the data are presented asmean±SD.

DPPH scavenging assay—The abilities of the polyphenols described hereinto scavenge the DPPH radical were measured optically by monitoring thedecreases of their absorptions at 429 nm. The DPPH scavenging activitiesof DHNB, DH6NB, DHBA, DHB-CHO, THB-CHO, and allopurinol were assayed ata concentration of 20 μM (FIG. 7A). DPPH was used at a concentration of100 μM. Their scavenging activities were compared with that of vitaminC. As shown in FIG. 7A, DHNB, DH6NB, DHBA, DHB-CHO, and THB-CHO have asstrong of a DPPH scavenging effect as vitamin C; however, allopurinolhas no scavenging effect on DPPH. The concentration dependent effects ofcompounds DHNB, DHBA, DHB-CHO, and allopurinol on DPPH scavengingactivity were also studied and compared with that of vitamins C and E(FIG. 8).

HOCl scavenging assay —HOCl was prepared immediately before use byadjusting the pH of a 1% (v/v) solution of NaOCl to pH 6.2 with 0.6 Msulfuric acid. The concentration was further determinedspectrophotometrically at 235 nm using the molar extinction coefficientof 100 M⁻¹ cm⁻¹. 5-Thio-2-nitrobenzoic acid (TNB) was prepared byreducing 5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB) with sodiumborohydride in phosphate buffer. The final concentrations of reagentsused in the assay are as follows: 25 μM HOCl, 70 μM TNB, 0 to 200 μMantioxidants, phosphate buffer, 50 mM, pH 6.6. The HOCl scavenging assaywas based on the inhibition of TNB oxidation to DTNB induced by HOCl.

At 20 μM, THB-CHO had a stronger HOCl scavenging effect than that ofvitamin C. DHNB had a moderate scavenging effect, while other compounds,including DHBA, DHB-CHO, and DH6NB, had a weak scavenging effect on HOCl(FIG. 7B). The concentration dependent effect of these compounds,including DHNB, caffeic acid, DHBA, DHB-CHO, DHB-COOH, and allopurinol,on HOCl scavenging activity were also studied and compared with that ofVitamin C (FIG. 9).

Peroxynitrite scavenging assay—Peroxynitrite (ONOO⁻) was generated bymixing 5 mL acidic solution (0.6 M HCl) of H₂O₂ (0.7 M) and 5 mL of 0.6M KNO₂ in an ice bath for 1 second and the reaction was quenched with 5mL of ice-cold 1.2 M NaOH. Residual H₂O₂ was removed using granular MnO₂prewashed with 1.2 M NaOH and the reaction mixture was then leftovernight at −20° C. Concentrations of ONOO⁻ were determined before eachexperiment at 302 nm using a molar extinction coefficient of 1,670 M⁻¹cm⁻¹. The final concentrations of reagents used in the assay are asfollows: 25 μM ONOO⁻, 10 μM DTPA, 5 μM DHR 123, 0.1M phosphate buffer,pH 7.4. The ONOO⁻ scavenging assay was performed by monitoring theoxidation of dihydrorhodamine (DHR 123) by ONOO⁻ spectrophotometricallyat 500 nm.

The abilities of DHNB, DHBA, DHB-CHO, DH6NB, caffeic acid, THB-CHO,gallic acid, vanillin, and DMB-CH₂OH to scavenge peroxynitrite werecompared with that of vitamin C. DHNB, DHBA, DHB-CHO, DH6NB, caffeicacid, THB-CHO and gallic acid had a strong scavenging effect on ONOO⁻(FIG. 7C). Vitamin C was used as a positive control. The concentrationdependent effects of these compounds on ONOO⁻ scavenging were alsostudied and compared with that of vitamins C and E (FIG. 10).

Superoxide scavenging assay—Superoxide (O₂ ^(−)) scavenging activitywas assayed in the xanthine-xanthine oxidase system and determined bythe inhibition of the reduction of nitro blue tetrazolium (NBT) to formblue formazan which has an absorption at 560 nm. The finalconcentrations of reagents used in the assay are as follows: 16.8 mUxanthine oxidase, 25 μM xanthine, 50 μM NBT, and 0.1M phosphate buffer(pH 8.5). O₂ ^(−) production and xanthine oxidase activity weremeasured as NBT reduction (at 560 nm) and uric acid production (at 295nm), respectively. The abilities of polyphenols to scavenge O₂ ^(−)were compared with that of GSH.

DHBA and THB-CHO, at the concentration of 20 μM, had strong scavengingeffects on superoxide (FIG. 7D). DHBA had an even stronger superoxidescavenging effect than that of glutathione. The concentration dependenteffect of DHBA and caffeic acid on superoxide scavenging was alsostudied and compared with that of glutathione (FIG. 11).

Antioxidant activity of Polyphenols—Several of the compounds describedherein strongly scavenged DPPH, ONOO⁻, HOCl, and superoxide ion with lowIC₅₀ values (see FIG. 7). Allopurinol does not possess the antioxidantproperties similar to the compounds described herein. Thus, theantioxidant properties of the compounds described herein are anadvantage as XO inhibitors over allopurinol.

Example 4 Hypouricemic Effect of DHNB in Allantoxanamide InducedHyperuricemic Mice

A hyperuricemia mouse model was used. Allantoxanamide, a potent uricaseinhibitor, was used to induce hyperuricemia in mice in this study.Briefly, adult C57BL/6 mice (15-25 g, 6-8 weeks old, 6 per group) wereadministrated DHNB at a concentration of 100 mg/kg in 1.0% polyethyleneglycol 400 (PEG400 in a volume of 0.1 ml/10 g mouse body weight) viaoral gavage. The mice were subsequently intraperitoneally injected withallantoxanamide at 200 mg/kg in 0.5% CMC-Na in a volume of 0.1 ml/10 gmouse body weight just after the tested drug oral administration toincrease the serum uric acid level. Positive control mice wereadministered allopurinol at the same concentration as DHNB followed byi.p. allantoxanamide. The negative control mice were administered PEG400only followed by i.p. allantoxanamide. The normal group mice wereadministered PEG400 only followed by i.p. CMC-Na only. Food and waterwere withheld overnight prior to the study. Whole blood samples werecollected from mice through orbital vein bleeding at the end of thestudy. The mice were anaesthetized with diethyl ether inside a chamber.The blood was allowed to clot for 1 h at room temperature and thencentrifuged at 2350×g for 4 min to obtain the serum. The serum was kepton ice and assayed immediately. Serum uric acid was determined with thephosphotungstate method, as known to those of skill in the art.

Both allantoxanamide and potassium oxonate have been used as uricaseinhibitors; however, the hyperuricemic effects of allantoxanamide arestronger and last longer than that of oxonate in rats. A singleintraperitoneal injection of 200 mg/kg allantoxanamide in miceprogressively increased the serum acid level during the experiment for 4hours. The serum urate levels were elevated from 2 mg/dl (normal mice)to 5.4, 9.5, 13.3, and 16.4 mg/dl in 1, 2, 3, and 4 h after theallantoxanamide i.p. injection, respectively. In contrast, when the micewere orally administered 100 mg/kg DHNB before the allantoxanamideinjection, the serum urate levels were significantly lowered in 2 hoursand maintained at a level just slightly higher than the normal level in4 hours. In comparison, when allopurinol was used in the same conditionas that of DHNB, allopurinol also significantly lowered the serum uratelevel close to the normal level in mice. See FIG. 12 and FIG. 13.

Example 5 Acute Toxicity Studies of DHNB in Mice

To determine whether DHNB has any acute toxicity in mice, C57BL/6 micewere randomized into 3 groups (12/group). Groups 1 to 3 received an oralvehicle solution (PEG400), DHNB (500 mg/kg), and allopurinol (500mg/kg), respectively. Each mouse was monitored for general healthconditions on a daily basis for 28 days, including examination ofmortality, body weights, and behavior of the mice.

Toxic effects of DHNB are not reported, but the lowest published lethaldose of DHNB in the mouse is 312 mg/kg (oral administration once). DHNBor allopurinol at 500 mg/kg was administrated to 12 mice, respectively,via oral gavage. Control mice received the vehicle solution. The animalswere observed daily up to 28 days. DHNB-treated mice did not show anysymptoms of general toxicity. There was no difference in body weight andbehavior between DHNB-treated mice and control mice. Histology analysisfor the liver, kidney, and heart did not show any difference betweenDHNB-treated mice and control mice. In the allopurinol treated mice,however, 5 mice died within 3 days (mortality 42%). Furthermore, thesurviving mice (mixed male and female) gave birth to total 19 pups, buteight died in two days. The survived pups of allopurinol treated micestarted to lose hair after two weeks (FIG. 14A) and lost most of theback hair at 3 weeks (FIG. 14B) to 4 weeks (FIG. 14C). After separatedfrom the adult mice, the pups started to grow hair again and returned tonormal hair at the age of 6 to 7 weeks (FIG. 14D). However, this hairloss phenomenon was not observed on DHNB treated mice (see FIG. 14E forDHNB treated mice at 4 weeks). A summary of the in vivo toxicities ofDHNB and allopurinol in mice is shown in Table 1.

TABLE 1 2^(nd) Generation Mortal- Mortal- 12 Mice/group Behavior Organsity ity Hair Loss DHNB 100 Normal Normal None N/A None mg/kg 200 NormalNormal None N/A None 500 Normal Normal None None None Allopurinol NormalN/A Average Average 1^(st) batch, 100% 500 mg/kg for 42% 42% 2^(nd)batch, 50% survivors 3^(rd) batch, 20%

Example 6 XO Inhibition Assay

XO inhibition assays were performed on the compounds shown below inScheme 2 using the methods as described in Example 1.

The inhibitory effects of DNSA and NHBA on XO activity were determinedby measuring the initial rate of formation of uric acid. Followingexposure of XO (5 milliunits/ml) to a 0-10 μM concentration of DNSA or a0-40 μM concentration of NHBA in 33 mM phosphate buffer (pH 7.4, 25°C.), XO activity was determined by the production of uric acid (295 nm).Reactions were initiated by the addition of xanthine (50 μM). Theresults are shown in FIGS. 15A and 15B.

The XO inhibition effects of the compounds shown in Scheme 2 werecompared at a concentration of 20 μM. XO activity was determined bymeasuring the initial rate of formation of uric acid (λ=295 nm) as inFIGS. 15A-B. After pre-incubation of 20 nM XO and 20 μM inhibitor for 10min, 50 μM xanthine was added to initiate the reaction. Data representthe mean±S.E. of at least three independent determinations and are shownin FIG. 16.

The influence of pre-incubation of DNSA with XO on the XO activity wasdetermined. XO activity was determined by the steady-state rate offormation of uric acid (λ=295 nm) by pre-incubation of 20 nM XO and 20μM DNSA for 0-10 min followed by the addition of 50 μM xanthine to startthe reaction. Data represent one of three independent determinations forDNSA. Pre-incubation of DNSA with XO strongly inhibited XO activity. Theresults are shown in FIG. 17.

The XO activities after pre-incubation with DNSA and DHNB for 20 h weredetermined. XO stored at room temperature for 20 h decayed in 30%activity, but XO/DNSA (5 μM or 10 μM) samples showed no XO activity atall and DNSA was not converted. For XO/DHNB samples, XO activity wasrecovered, and DHNB was converted to DHNB-COOH completely. The resultsare shown in FIG. 18.

The compounds and methods of the appended claims are not limited inscope by the specific compounds and methods described herein, which areintended as illustrations of a few aspects of the claims and anycompounds and methods that are functionally equivalent are within thescope of this disclosure. Various modifications of the compounds andmethods in addition to those shown and described herein are intended tofall within the scope of the appended claims. Further, while onlycertain representative compounds, methods, and aspects of thesecompounds and methods are specifically described, other compounds andmethods and combinations of various features of the compounds andmethods are intended to fall within the scope of the appended claims,even if not specifically recited. Thus, a combination of steps,elements, components, or constituents may be explicitly mentionedherein; however, all other combinations of steps, elements, components,and constituents are included, even though not explicitly stated.

1-15. (canceled)
 16. A method for treating gout or hyperuricemia in asubject comprising administering to the subject an effective amount of acompound comprising:

or a pharmaceutically acceptable salt thereof, wherein: R¹, R², R³, R⁴,and R⁵ are each independently selected from hydrogen, hydroxyl, cyano,fluoro, chloro, bromo, trifluoromethyl, sulfonyl, and aldehyde, whereinR¹, R², R³, R⁴, and R⁵ are not simultaneously hydrogen.
 17. The methodof claim 16, wherein the compound is selected from the group consistingof:


18. The method of claim 16, further comprising administering a secondtherapeutic agent to the subject.
 19. The method of claim 16, whereinthe second therapeutic agent is an anti-gout agent.
 20. The method ofclaim 18, wherein the anti-gout agent is selected from the groupconsisting of allopurinol, benzbromarone, colchicine, probenecid, andsulfinpyrazone.
 21. The method of claim 16, wherein the secondtherapeutic agent is an anti-inflammatory agent.
 22. The method of claim16, wherein the second therapeutic agent is an antioxidant.
 23. Themethod of claim 16, wherein the compound is administered orally.